Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of HA crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles (MVs). Our working model is that bone mineralization is first initiated within the lumen of MVs. In a second step, HA crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu where they continue to propagate along collagen fibrils. Our recent data have indicated that tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role in restricting the concentration of extracellular inorganic pyrophosphate (PP), a mineralization inhibitor, to maintain a P/PPi ratio permissive for normal bone mineralization. Using a variety of single and double gene knockout experiments we have found that mice deficient in TNAP function, i.e., Akp2-/- mice, display osteomalacia due to an arrest in the propagation of HA crystals outside the MVs caused by an increase in extracellular PPi concentrations. Inside the MVs, however, HA crystals are still present in Akp2-/- mice. We hypothesize that a newly identified soluble phosphatase, PHOSPHO1, with specificity for phosphoethanolamine (PEA) and phosphoserine (PS) present in the MVs, is responsible for increasing the local concentration of Pi inside the MVs to change the P/PPi ratio to favor precipitation of HA seed crystals. We will test this hypothesis by affecting the first and second steps of MV-mediated mineralization using a genetic and pharmacological approach. Experimentally we will characterize the mineralization abnormalities and related metabolic changes in mice deficient in Phospho1 expression compared to Akp2-/- mice and assess the effect of the simultaneous inactivation of the Phospho1 and Akp2 genes on skeletal mineralization. We will also study the effects of ablating or inhibiting PHOSPHO1 and/or TNAP activity on the ability of osteoblast-derived MVs to initiate and propagate calcification in vitro. We have now established an assay and screened the LOPAC and the Spectrum libraries and identified 17 compounds capable of inhibiting recombinant PHOSPHO1 activity with IC50 values of 10 [unreadable]M or less. Three of these 17 compounds were selected for further characterization, i.e., SCH 202676; Lansoprazole and Ebselen. Lansoprazole and SCH 202676 decreased the amount of liberated Pi using isolated osteoblast-derived MV further indicating the involvement of PHOSPHO1 in the initiation of skeletal calcification. In response to this "Solicitation of Assays for High Throughput Screening (HTS) in the Molecular Libraries Screening Centers Network, PAR-06-545, we are submitting this R03 proposal to screen additional libraries in order to identify/develop the best possible PHOSPHO1-specific inhibitors to help us clarify the role of this enzyme in skeletal mineralization. [unreadable] [unreadable] [unreadable]